Polypeptide Purification

Abstract

The invention relates to the purification of different gamma carboxylated forms of a polypeptide using ion exchange chromatography. In particular, the invention provides a method for purifying a polypeptide having a desired content of gamma-carboxyglutamic acid from a sample comprising mixture of species of said polypeptide having different contents of gamma-carboxyglutamic acid, said method comprising the steps of: (a) loading said sample onto an anion exchange chromatography material; (b) eluting said polypeptide using a solution at a pH of less than pH 9.0 comprising at least one salt selected from ammonium acetate, ammonium chloride and sodium acetate; and (c) selecting a fraction obtained from said elution wherein the polypeptides in the fraction have the desired content of gamma-carboxyglutamic acids.

1 . A method for purifying a polypeptide having a desired content of gamma-carboxyglutamic acid from a sample comprising mixture of species of said polypeptide having different contents of gamma-carboxyglutamic acid, said method comprising the steps of:
 (a) loading said sample onto an anion exchange chromatography material;   (b) eluting said polypeptide using a solution at a pH of less than pH 9.0 comprising at least one salt selected from ammonium acetate, ammonium chloride and sodium acetate; and   (c) selecting a fraction obtained from said elution wherein the polypeptides in the fraction have the desired content of gamma-carboxyglutamic acids.   
     
     
         2 . A method according to  claim 1  comprising the following steps:
 (a) equilibrating an anion exchange material with a buffer at a pH of less than 9.0; 
 (b) loading said sample onto the anion exchange material; 
 (c) optionally washing the anion exchange material with a buffer at a pH of less than 9.0; 
 (d) eluting said polypeptide from the ion exchange material using a solution at a pH of less than 9.0 comprising at least one salt selected from ammonium acetate, ammonium chloride and sodium acetate; and 
 (e) selecting a fraction obtained from said elution wherein the polypeptides in the fraction have the desired content of gamma-carboxyglutamic acids. 
 
     
     
         3 . A method according to  claim 1  wherein the polypeptide to be purified is selected from Factor IX, Factor VII, Factor VIIa, Factor X, Prothrombin, Protein-S, Protein-C, Protein Z, Osteocalcin, Matrix-gla-protein, Growth arrest-specific-6, Proline-rich-Gla-1, Proline-rich-Gla-2, Proline-rich-Gla-3 and Proline-rich-Gla-4. 
     
     
         4 . A method according to  claim 3  wherein the polypeptide is Factor IX. 
     
     
         5 . A method according to  claim 4  wherein said method comprises selecting a fraction obtained from said elution which has an increase in the proportion of #1-11-Gla and/or #1-12-Gla forms of Factor IX compared with the proportion of #1-11-Gla and/or #1-12-Gla forms of Factor IX in the sample being purified. 
     
     
         6 . A method according to  claim 4  wherein said method comprises selecting a fraction obtained from said elution which has an decrease in the proportion of #1-10-Gla form of Factor IX compared with the proportion of #1-10-Gla form of Factor IX in the sample being purified 
     
     
         7 . A method according to  claim 3  wherein the polypeptide is Factor VII or Factor VIIa. 
     
     
         8 . A method according to  claim 7  wherein said method comprises selecting a fraction obtained from said elution which has an increase in the proportion of #1-10-Gla and/or #1-11-Gla forms of Factor VII or Factor VIIa compared with the proportion of #1-10-Gla and/or #1-11-Gla forms of Factor VII or Factor VIIa in the sample being purified. 
     
     
         9 . A method according to  claim 7  wherein said method comprises selecting a fraction obtained from said elution which has an decrease in the proportion of #1-9-Gla form of Factor VII or Factor VIIa compared with the proportion of #1-9-Gla form of Factor VII or Factor VIIa in the sample being purified. 
     
     
         10 . A method according to  claim 1  wherein said elution buffer has a pH of between 5.0 and 8.5. 
     
     
         11 . A method according to  claim 1  wherein ammonium acetate, ammonium chloride or sodium acetate is present in the elution buffer at between 0.1M and 2.0M 
     
     
         12 . A method according to  claim 11  wherein ammonium acetate, ammonium chloride or sodium acetate is present in the elution buffer at about 0.6 M. 
     
     
         13 . A method according to  claim 1  wherein said ion exchange chromatography utilises an equilibration buffer at a pH between 5.0 and 8.5. 
     
     
         14 . A polypeptide formulation obtained by a method according to  claim 1 .